The present invention relates to novel plant chimeric promoters that are responsive to sugar depletion conditions in a plant cell.
The Amy3D and Amy3E genes are two sugar-regulated genes in the rice xcex1-amylase family (Huang, N. et al., Nucleic Acids Res., 18(23):7077 (1990)). Amy3D is expressed in seedlings in response to sugar depletion and is not expressed when sugar, such as glucose, concentrations are elevated. Further, Amy3D is upregulated strongly in cell culture under conditions of sugar depletion or sugar deprivation. Unlike many other xcex1-amylase genes, Amy3D is not induced in response to gibberellic acid.
Metabolic regulation of Amy3D expression provides a signal mechanism to help control sugar production in the source tissues of germinating cereal,seedling. Thus, the rate of starch breakdown is modulated in response to the rate at which the embryo axis can utilize sugar for its growth.
In one aspect, the invention includes a chimeric plant promoter for upregulating expression of a coding sequence operatively linked to the promoter, under conditions of sugar depletion or deprivation in a plant cell. The promoter includes a promoter element effective to express the coding sequence, under selected conditions and, carried in the promoter element, one or more of the following heterologous sequences which are responsive to (upregulated by) sugar depletion in plant cells: SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, and combinations of these sequences. An exemplary combination is produced from sequences from SEQ ID NOS: 2 and 3, identified as SEQ ID NO:5.
In one embodiment the heterologous sequences is duplicated. In another embodiment, the promoter element is a monocot amylase-gene promoter, and the heterologous sequence is inserted in the promoter element at a position homologous to the position of the heterologous sequence in the rice Amy3D gene. Exemplary promoter elements are from the RAmy1A, RAmy1B, RAmy2A, RAmy3A, RAmy3B, RAmy3C, pM/C, gKAmy141, gKAmy155, Amy32b, and HV18 genes.
Also forming part of the invention is a method of enhancing the inducibility by sugar depletion or deprivation of a plant or plant-virus promoter in plant cells. The method includes introducing into a plant promoter or plant-virus promoter, one or more of the above heterologous sequences, to achieve at least a 2-fold greater sugar-depletion induction level over that of the unmodified promoter.
The promoter may be, for example, an xcex1-amylase promoter from a monocot xcex1-amylase gene, such as the RAmy1A, RAmy1B, RAmy2A, RAmy3A, RAmy3B, RAmy3C, pM/C, gKAmy141, gKAmy155, Amy32b, or HV18 genes. The promoter may be duplicated in the chimeric promoter. The plant may be a monocot plant.
In another aspect, the invention includes a vector for use in transforming a plant. The vector includes a chimeric gene having, operatively linked in sequence in a 5xe2x80x2 to 3xe2x80x2 direction, (i) the above chimeric plant promoter, (ii) a gene encoding a protein to be expressed, and (iii) a 3xe2x80x2 untranslated terminator region. Also disclosed are plant cells transfected with the vector.